Until fairly recently, most research and applications relating to DNA have been concerned with the conventional Watson-Crick structure wherein two helical polynucleotide strands form a duplex because of hydrogen bonding between the bases on one strand to those of the other strand to form the purine-to-pyrimidine base pairs, AT and GC.
It has been known for some time that the polynucleotide polydT will bind to the polydA-polydT duplex to form a collinear triplex (Arnott, S & Selsing E. (1974) J. Molec. Biol. 88, 509). The structure of that triplex has been deduced from X-ray fiber diffraction analysis and has been determined to be a collinear triplex (Arnott, S. & Selsing E. (1974) J. Molec. Biol. 88, 509). The polydT strand is bound in the parallel orientation to the polydA strand of the underlying duplex. The polydT-polydA-polydT triplex is stabilized by T-A Hoogstein base pairing between A in the duplex and the third strand of polydT. That interaction necessarily places the third strand, called a ligand, within the major groove of the underlying duplex. The binding site in the major groove is also referred to as the target sequence.
Similarly, it has been shown that polydG will bind by triplex formation to the duplex polyG-polydC, presumably by G--G pairing in the major helix groove of the underlying duplex, (Riley M., Mailing B. & Chamberlin M. (1966) J. Molec. Biol. 20, 359). This pattern of association is likely to be similar to the pattern of G-G-C triplet formation seen in tRNA crystals (Cantor C. & Schimmel P., (1980) Biophysical Chemistry vol. I, p. 192-195).
Triplexes of the form polydA-polydA-polydT and polydC-polydG-polydC have also been detected (Broitman S., Im D. D. & Fresco J. R. (1987) Proc. Nat. Acad. Sci U.S.A. 84, 5120 and Lee J. S., Johnson D. A. & Morgan A. R. (1979) Nucl. Acids Res. 6, 3073). Further the mixed triplex polydCT-polydGA-polydCT has also been observed. (Parseuth D. et al. (1988) Proc. Nat. Acad Sci. U.S.A. 85, 1849 and Moser H. E. & Dervan P. B. (1987) Science 238,645). These complexes, however, have proven to be weak or to occur only at acid PH.
Parallel deoxyribo oligonucleotide isomers which bind in the parallel orientation have been synthesized (Moser H. E. & Dervan P. E. (1987) Science 238, 645-650 and Rajagopol P. & Feigon J. (1989) Nature 339, 637-640). In examples where the binding site was symmetric and could have formed either the parallel or antiparallel triplex (oligodT binding to an oligodA-oligodT duplex target), the resulting triplex formed in the parallel orientation (Moser H. E. & Dervan P. E. (1987) Science 238, 645-650 and Praseuth D. et al (1988) PNAS 85, 1349-1353), as had been deduced from x-ray diffraction analysis of the polydT-polydA-polydT triplex.
Studies employing oligonucleotides comprising the unnatural alpha anomer of the nucleotide subunit, have shown that an antiparallel triplex can form (Praseuth D. et al. (1988) PNAS 85, 13449-1353). However, since the alpha deoxyribonucleotide units of DNA are inherently reversed with respect to the natural beta subunits, an antiparallel triplex formed by alpha oligonucleotides necessarily follows from the observation of parallel triplex formation by the natural beta oligonucleotides. For example, alpha deoxyribo oligonucleotides form parallel rather than antiparallel Watson-Crick helices with a complementary strand of the beta DNA isomer.
It has been demonstrated that a DNA oligonucleotide could bind by triplex formation to a duplex DNA target in a gene control region; thereby repressing transcription initiation (Cooney M. et al. (1988) Science 241, 456). This was an important observation since the duplex DNA target was not a simple repeating sequence.
U.S. Pat. No. 5,176,996 issued on Jan. 5, 1993 to Hogan et al. discloses a method for making synthetic oligonucleotides which bind to target sequences in a duplex DNA forming collinear triplexes by binding to the major groove. This method includes scanning genomic duplex DNA and identifying nucleotide target sequences of greater than about 20 nucleotides having either about at least 65% purine bases or about 65% pyrimidine bases; and synthesizing synthetic oligonucleotides complementary to identified target sequences. The synthetic oligonucleotides have a T when the complementary location in the DNA duplex has a GC pair and have a 7 when the complementary location in the DNA duplex has an AT basepair. These synthetic oligonucleotides are oriented 5' to 3' and bind parallel or 3' to 5' and bind antiparallel to the about at least 65% purine strand.
DNA triple helices have been reported in the literature to have applications in inhibiting and regulating the function of targeted genes. For example, McShan et al, J. Biol. Chem., 267, 5712, (1992), reported that mixed purine-pyrimidine oligodeoxyribonucleotides designed to form collinear DNA triplexes with purine-rich elements in the HIV-1 promoter inhibit the transcription of HIV-1 in infected human cells.
Also, Postel et al, Proc. Nat'l Acad. Sci., 88, 8227, (1991) reported a triplex-forming oligonucleotide which binds to the C-myc promoter in HeLa cells and inhibits the transcription of C-myc mRNA. Further, Weiss et al, Abstract, Int. Conf. on Nucl. Acid Med. Appl., Abstract 4-34, demonstrate that a 26mer designed to form a triple helix with an interferon inducible gene caused a reduction in gene expression from HeLa cells, keratinocytes, corneal cells and retinal pigmented endothelial cells in a dose dependent manner at micromolar concentrations. However, there still exists a need in the art for polynucleotide sequences having unique structures and properties.